In many applications it is desirable to enrich, or alternatively deplete, certain cell populations in a biological sample. For example, the separation of specific cell types from peripheral blood, bone marrow, spleen, thymus and fetal liver is key to research in the fields of haematology, immunology and oncology, as well as diagnostics and therapy for certain malignancies and immune disorders.
Most cell separation techniques require that the input sample be a single cell suspension. For this reason, blood has historically been the most common tissue used for cell separations. Purified populations of immune cells such as T cells and antigen presenting cells are necessary for the study of immune function and are used in immunotherapy. Investigation of cellular, molecular and biochemical processes requires analysis of certain cell types in isolation. Numerous techniques have been used to isolate T cell subsets, B cells, basophils, NK cells and dendritic cells from blood for these investigations.
More recently, enzymatic digestion methods have been developed to dissociate tissues from solid organs into single cell suspensions, permitting distinct cell types to be isolated. This is of particular benefit to the study of pluripotent stem cells and tissue-specific stem cells from adults. The rapidly growing field of stem cell research is spurred by the potential of these cells to repair diseased or damaged tissues. Bone marrow (hematopoietic) stem cells were the first adult stem cells to be purified and used clinically and the therapeutic potential of hematopoietic stem cells is now well documented. Transplantation of hematopoietic cells from peripheral blood and/or bone marrow is increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of a variety of disorders including malignant, nonmalignant and genetic disorders. Very few cells in such transplants are capable of long-term hematopoietic reconstitution, and thus there is a strong stimulus to develop techniques for purification of hematopoietic stem cells. Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogenic grafts, and tumor cells in autologous transplants that may cause recurrence of the malignant growth. It is also important to debulk the graft by removing unnecessary cells and thus reducing the volume of cyropreservant to be infused.
In certain instances it is desirable to remove or deplete tumor cells from a biological sample, for example in bone marrow transplants. Epithelial cancers of the bronchi, mammary ducts and the gastrointestinal and urogenital tracts represent a major group of solid tumors seen today. Micrometastatic tumor cell migration is thought to be an important prognostic factor for patients with epithelial cancer (Braun et al., 2000; Vaughan et al., 1990). The ability to detect such metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods. A technique to enrich circulating epithelial tumor cells in blood samples would increase the ability to detect metastatic disease and facilitate the study of such rare cells to determine the biological changes which enable spread of the disease.
Hematopoietic cells and immune cells have been separated on the basis of physical characteristics such as density and on the basis of susceptibility to certain pharmacological agents which kill cycling cells. The advent of monoclonal antibodies against cell surface antigens has greatly expanded the potential to distinguish and separate distinct cell types. There are two basic conceptual approaches to separating cell populations from blood and related cell suspensions using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labeled with the antibody(s).
In positive selection techniques the desired cells are labeled with antibodies and removed from the remaining unlabeled/unwanted cells. In negative selection, the unwanted cells are labeled and removed. Antibody/complement treatment and the use of immunotoxins are negative selection techniques, but FACS sorting and most batch-wise immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques, cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e.g. column of beads, flask, magnetic particles. Immunoadsorption techniques have won favour clinically and in research because they maintain the high specificity of cell targeting with monoclonal antibodies, but unlike FACSorting, they can be scaled up to directly process the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins and complement. They do however, require the use of a “device” or cell separation surface such as a column of beads, panning flask or magnet.
Current techniques for the isolation of hematopoietic stem cells, immune cells and circulating epithelial tumor cells all involve an initial step to remove red cells prior to antibody mediated adherence to a device or artificial particle (Firat et al., 1988; de Wynter et al., 1975; Shpall et al., 1994; Thomas et al., 1994; Miltenyi Biotec Inc., Gladbach, Germany). In the case of positive selection there is yet another step; removal of the cells from the device or particle. These multiple steps require time and incur cell loss.
Discontinuous density gradient centrifugation is commonly used to isolate peripheral blood mononuclear cells from granulocytes and erythrocytes. FICOLL-PAQUE®, a solution of Ficoll400 and diatrizoate sodium with a density of 1.077 g/ml, (Amersham Pharmacia Biotech AB, Uppsala Sweden) is one of the most popular density separation solutions used for this application. In a Ficoll density separation whole blood is layered over Ficoll, and then centrifuged. The erythrocytes, granulocytes and approximately 50% of the mononuclear cells settle to the cell pellet while the remaining 50% of the mononuclear cells settle to the Ficoll plasma interface. The success of this technique relies on the difference in density between mononuclear cells and granulocytes/erythrocytes as well as the choice of the density separation medium (DSM). During centrifugation, cells that are more dense than the DSM settle through the DSM forming a pellet at the bottom of the tube, while cells that are less dense than the DSM collect at the interface between the DSM and the cell suspension medium (e.g. plasma in the case of peripheral blood, cell culture medium in the case of cultured cells or dissociated tissue cells). Multiple layers of DSM having different densities can be used to divide the cells into multiple fractions. The same effect can be achieved by centrifuging cells in a medium with a continuous density gradient and then collecting the cells from different positions in the gradient. Sedimentation rate can also be used to separate cells of different density, but the separation time is Influenced not only by density but also the viscosity of the suspension and the cell size.
All density separation techniques have the same basic limitation; they can not separate subpopulations of cells with overlapping density distributions such as human lymphocyte subsets. Simple density separation techniques do not offer the high cell specificity offered by antibody-mediated techniques. To address this, dense particles have been targeted to cells using monoclonal antibodies with affinity to cells surface antigens and used in discontinuous or continuous density gradient centrifugation to separate cell populations with similar densities (Bildirici and Rickwood, 2001; Bildirici and Rickwood, 2000; Patel and Rickwood, 1995; Patel et al. 1993; U.S. Pat. No. 5,840,502; and StemCell Technologies, Supplement to 1999/2000 Catalogue). There are two advantages of using a discontinuous density gradient rather than a continuous density gradient in dense particle mediated cell separation. Discontinuous gradients are easier to prepare and offer a visible boundary (interface) where cells not bound to particles selectively collect.
Several patents (U.S. Pat. No. 5,840,502, U.S. Pat. No. 5,648,223, U.S. Pat. No. 5,646,004 and U.S. Pat. No. 5,474,687) describe the use of dense particles for negative selection by selectively targeting and pelleting undesired cell types using discontinuous density gradient separations. These patents state that the optimum density of the DSM for dense particle separation is within ±0.0005 to ±0.0002 g/cm3 of the density of the desired cell population.
There are no documented techniques for positive selection of cells using dense particles and discontinuous density gradients. In positive selection the desired cells are targeted with antibodies and dense particles and pelleted during separation.